Supernatants derived from primary as well as transformed CD8+ cells have potent HIV inhibitory activity. Not all of that activity can be accounted for by known active components including 2-chemokines. Using a strategy designed to screen specifically for novel factors that inhibit HIV in primary macrophages following viral entry we identified and have reported that prothymosin alpha (ProT1), a protein found in the cell-culture media of the HVS-transformed CD8+ T-cell-line, K#1 50K, has potent HIV-1 inhibitory activity (41). Depletion of native ProT1 from an HIV-1 inhibitory fraction of CD8+ cell supernatants removes the inhibitory activity, supporting its role in inhibition via soluble mediators. ProT1 is an abundant, acidic peptide that has been reported to be localized in the nucleus and associated with cell proliferation and activation of transcription. ProT1 suppresses HIV-1 replication, its activity is target-cell specific and inhibition predominantly occurs following viral integration (41). Native and recombinant ProT1 protein potently inhibit HIV-1 LTR-driven gene expression in macrophages and dendritic cells. The mechanism(s) of ProT1 -mediated suppression of integrated HIV-1 is not yet known. Our hypothesis is that exogenous ProT1 interacts with a cell surface receptor triggering signal transduction pathways that inhibit HIV-1 gene expression. The cell specificity of the effect is due to the involvement of cell specific transcription factors in HIV replication. In order to design an approach to understanding the mechanism of the post integrational suppression of HIV-1 by ProT1 some initial questions need to be addressed as outlined in this R-21 proposal. These include the relationship between activity and cellular uptake/nuclear localization, the role of major signaling pathways in the observed inhibition and the post-integration step in the HIV life cycle that is inhibited. Answers to these basic questions will allow the rational design of an experimental approach to determine the mechanism of action of this host restriction pathway. This initial approach will take advantage of what has been determined in other systems regarding function and functional domains of ProT1. Understanding the mechanism of suppression of proviral HIV-1 by ProT1 and identification of the active anti-HIV-1 domain(s) of this molecule will open new avenues for developing therapeutics targeting integrated HIV-1.After the human immunodeficiency virus type-1 (HIV-1) infects cells of the immune system the viral genome becomes integrated into the host genome. Viral replication at the post-integration step of the viral life cycle leads to production of new viral particles that are shed from the infected cell and go on to infect other cells. This is of particular importance with infected macrophages which can shed viral particles for up to 2-3 month. Post-integration events in HIV-1 life cycle are difficult to interrupt because there are very few drugs able to interfere with this part of the viral life cycle. We have recently shown that the human protein prothymosin alpha (ProT1) very effectively interferes with HIV-1 at the post-integration step of its life cycle leading to decrease of viral production. Investigation of the mechanism of action of ProT1 in HIV-1 suppression will lead to the development of therapeutics that can block this important step of the viral life cycle.